The present invention relates to chromogenic enzyme substrate compounds which are useful as optical indicator compounds in analytical test systems. In particular, the present invention relates to chromogenic .alpha.-amylase substrate compounds for the detection of .alpha.-amylase in a liquid test sample.
.alpha.-Amylase is an endoenzyme which hydrolyzes complex carbohydrates, such as starch and glycogen, which are polymers of .alpha.-D-glucose units linked through the 1 and 4 carbon atoms located on adjacent glucose residues. Although there are many amylase isoenzymes present in serum and urine, the predominant species are from the pancreas (p-type amylase) and salivary glands (s-type amylase), with the p-type comprising approximately one-half of the total amylase activity. During certain disease states, such as acute pancreatitis and salivary lesions, the level of amylase in serum and urine becomes increasingly elevated. Accordingly, the determination of total amylase activity in serum or urine is particularly useful for the clinical diagnosis of pancreatitis and mumps.
A number of methods have been developed over the years to make such determination, including the measurement of the turbidity or viscosity of an aqueous starch solution or the amount of reducing sugar formed in the reaction [Henry, et al., Clin. Chem., vol. 6, p. 434 (1960) and Ware, et al., Standard Method of Clinical Chemistry, Vol. 4, p.15 (1963)], as well as kinetic methods [Barbson, et al., Clin. Chem., Vol. 14, p. 802 (1968), Reinderknecht, et al., Experientia, Vol. 23, p. 805 (1967), and Klein, et al., Anal. Biochem., Vol. 31, p. 412 (1960)]. Recently, methods have been developed which employ maltooligosaccharides or glucosides as .alpha.-amylase substrates whereby glucose or maltose produced by the action of .alpha.-amylase is determined by coupled enzyme systems [Lalegeri, et al., Clin. Chem., Vol. 28, p. 1798 (1982), Hagele, et al., J. Chromatog., Vol. 223, p. 69 (1981), Kaufman, et al., Clin. Chem., Vol. 26, p. 846 (1980) and Pierri, et al., Methods in Enzymatic Analysis, Vol. 4, p. 146 (1984)]. However, such assays require the elimination of interferants such as endogenous glucose and maltose present in a test sample before the assay can be conducted.
More recently, chromogenic methods have been developed employing maltooligosaccharides coupled to a soluble dye or chromogen which provides a detectable optical signal when the chromogen is liberated. However, since .alpha.-amylase is capable of cleaving only covalent bonds between adjacent glucose units, it is necessary to employ an endoenzyme such as .alpha.-glucosidase or .beta.-glucosidase to cleave the bond between the final glucose unit and the chromogen coupled thereto. The liberated form of the chromogen has the desired optical activity which can be measured and correlated to the amount of .alpha.-amylase present in a liquid test sample. For example, U.S. Pat. No. 4,544,631 describes a chromogenic amylase assay using p-nitrophenyl-.alpha.-D-maltooligosaccharide, with four to seven glucose units, as the substrate. The p-nitrophenyl moiety is covalently bound to the reducing end of the maltooligosaccharide through a .alpha.-1,4-hemiacetal linkage whereby when hydrolyzed by amylase and an indicator enzyme, such as .alpha.-glucosidase, the rate of formation of yellow nitrophenol is used to quantitate amylase activity.
Typically, such chromogenic substrates for .alpha.-amylase are prepared employing cyclodextrin glucanotransferase (CDGT), derived from Bacillus macerans (EC 2.4.1.19), according to methods known in the art [French, et al., J. Am. Chem. Soc., Vol 76, p. 2387 (1954), Bender, Carbohydrate Research, Vol. 78, pp. 133 and 147 (1980), and Wallenfels, et al., Carbohydrate Research, Vol. 61, p.539 (1978)]. Such methods rely upon the known glycosyl-transferring properties of CDGT which involves the transfer of a glucoside compound to a pre-formed polysaccharide chain (e.g., .alpha.-cyclodextrin) by CDGT, i.e., transglucosylation, to result in the desired .alpha.-amylase substrate compound (e.g., maltoheptaoside). In addition to such transglucosylation property, CDGT is also capable of catalyzing the hydrolysis of long-chain maltooligosaccharides into shorter lengths thereof (e.g., glucose, maltosides, maltotriosides, and the like) which do not serve as substrates for .alpha.-amylase. Accordingly, it is therefore necessary to provide reaction conditions which maximize the synthetic activity of CDGT while, at the same time, minimize the hydrolytic activity thereof, in order to provide high yields of .alpha.-amylase substrate compounds having the required length of the maltooligosaccharide chain. The prior art methods previously described provide reaction conditions which result in low yields of the desired compound (e.g., maltoheptaoside or derivatives thereof), usually less than 5%, and typically require reaction times of 24 hours or more.
Accordingly, it is an object of the present invention to provide a convenient method for preparing chromogenic .alpha.-amylase substrate compounds at high preparative yields within a relatively short period of time.
Further, it is an object of the present invention to provide chromogenic .alpha.-amylase substrate compounds which are sensitive to the presence of .alpha.-amylase to thereby permit the rapid and accurate determination thereof in an analytical test system.